PROJECT SUMMARY Cocaine and alcohol are commonly abused and frequently co-abused drugs. Available medications do not meet the needs for treating ongoing cocaine and alcohol abuse, relapse and co-abuse. The modified human butyrylcholinesterase (hBChE) exhibits great catalytic potency and substrate specificity for cocaine hydrolysis and is effective in reducing the behavioral and toxic effects of cocaine in rodents. The glucagon-like peptide 1 (GLP1) receptor agonists can attenuate the reinforcing properties of cocaine and alcohol in rodents. Both hBChE and GLP1 have very short half-lives in vivo, however, limiting their potential in treating cocaine abuse and co-abuse with alcohol. Cultured epidermal autografts have been clinically used for treatment of massive skin wounds for decades. Transplantation of skin grafts derived from engineered skin progenitor cells provides a novel and ideal approach for long-term and efficient delivery of therapeutic agents in vivo. We have made key technical advancement in developing a novel mouse-to-mouse skin transplantation method that allows the stable introduction of engineered epidermal progenitor cells into immunocompetent host mice. We have also used the CRISPR technology to target either an hBChE gene or a doxycycline (dox)-inducible modified GLP1 gene (DImGLP1) into epidermal progenitor cells. We then transplanted the engineered hBChE or DImGLP1 cells into mice either separately (GhBChE and GDImGLP1) or together (GBChEGLP1). GhBChE and GDImGLP1 mice did not develop cocaine- and ethanol-induced conditioned place preference (CPP) respectively. Grafting hBChE cells right after CPP expression attenuated cocaine-induced reinstatement of CPP. GBChEGLP1 mice did not develop CPP and they exhibited a lower lethality rate induced by cocaine and ethanol co-administration than those in control mice. The goal of this proposal is to take advantage of these novel platforms to test an innovative cutaneous gene therapy for cocaine abuse and co-abuse with alcohol in mice. We will also evaluate duration of hBChE and mGLP1 protection against cocaine and/or alcohol abuse, potential immune responses and approaches to reduce them. We expect the proposed cutaneous gene therapy to be long-lasting, highly specific and efficient with little individual variation. To be able to engineer stable in vivo bio-delivery systems for therapeutic genes via epidermal progenitor cells is significant because it holds potential for reducing ongoing cocaine abuse and co-abuse with alcohol and relapse in users and addicts. The proposed work will have a high impact in that results will lay key groundwork for the development of a highly personalized, long-lasting and affordable approach for combating cocaine abuse and co-abuse with alcohol.